Method for the inactivation of amylase in the presence of protease

ABSTRACT

The invention relates to a method for the inactivation of amylase in a solution comprising  Rhizomucor miehei  aspartic protease by keeping the solution at a pH between 2.1 and 2.8 and at a temperature between 20° C. and 40° C. for a period of time sufficient to inactivate amylase for at least 95%. With this method the level of residual  Rhizomucor miehei  aspartic protease activity in the solution at the end of the heating time is at least 90%, preferably at least 95%, and the residual amylase activity is 0.05 RAU/g or lower, more preferably 0.001 RAU/g or lower, i.e. more than 99% of the amylase activity has been inactivated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage of PCT application PCT/EP02/14529having an international filing date of 18 Dec. 2002, which claimspriority from European application 01204991.2, filed 19 Dec. 2001. Thecontents of these documents are incorporated herein by reference.

This invention relates to a method for the inactivation of amylase inthe presence of protease. The invention further relates to a Rhizomucormiehei aspartic protease obtainable by the method of the invention andto its use in the making of dairy products.

Chymosin, extracted from the calf stomach is, at present, one of themost efficient milk-clotting enzymes known and is largely used in theproduction of cheese. However the increasing demand for chymosin by thedairy farming industry and its limited availability from natural sourceshas prompted scientists to search for suitable alternatives. Chymosincan now be produced via recombinant DNA technology in bacteria, i.e.Escherichia coli, yeast, i.e. Kluyveromyces lactis, and in filamentousfungi, i.e. Aspergillus niger. Also fungal proteases are now beingextensively used in cheese production. Rhizomucor miehei asparticprotease is the most common; milk-clotting preparation for its goodperformance (Sternberg, M. Z. (1971) Crystalline milk clotting proteasefrom Mucor miehei, and some of its properties. J. Dairy Sci., Vol. 54,page 159-167). This protease can also be produced heterologously viarecombinant DNA technology in filamentous fungi (Boel E. et al. EuropeanPatent EP-238023). Rhizomucor miehei aspartic protease is highly heatstable. It remains partially active in the whey after pasteurisation,causing undesired milk clotting when the whey is used in otherpreparations, like in enriched milk for baby food. Therefore, Rhizomucormiehei aspartic protease is often modified to decrease its heatstability. Several methods to increase thermal destabilisation ofRhizomucor miehei aspartic protease are known in the art. Some of themare described in the UK Patents GB 2038339B, GB 2045772B and GB2045773B.

A problem with the microbial production of milk-clotting enzymes viafermentation is the expression of other undesirable enzymes. Because ofan increasing demand in the market for pure enzyme preparations, severalmethods have been developed in the art for the separation andpurification of desirable enzymes from enzyme mixtures or for theselective inactivation of undesirable enzymes.

U.S. Pat. No. 2,683,682 (1954) discloses the differential inactivationof one of the enzymes of the group consisting of proteases and amylasesfrom mixtures comprising both enzyme types. The method comprisesadjusting the pH of the aqueous solution between 3.0 and 4.5 (toselectively inactivate amylase) or between 7.0 and 10.5 (to selectivelyinactivate protease) and maintaining the mixture at a temperaturecomprised between about 5° C. and 60° C. for a period of time sufficientto inactivate the undesired enzyme, generally from about 0.5 hours orlower for higher temperatures, to 20 hours for lower temperatures.Treatment of enzyme mixtures derived from malted wheat flour or maltedbarley at pH 3.6 and 50° C. for 0.5 hours leads to a protease recoveryof at best 66% and amylase deactivation of 99.7%. At the same pH and 5°C. for 20 hours the protease recovery is 85%, with an amylasedeactivation of 99.6%.

U.S. Pat. No. 4,086,139 (1978) describes the selective inactivation ofamylase in enzyme mixtures comprising protease and amylase. Theinactivation of amylase occurs by treating the enzyme mixture with anoxidising agent selected from the group consisting of chlorite andhypochlorite ions. The ions are added to the enzyme mixture insufficient amount to inactivate the amylase while leaving the proteaseintact, thus avoiding further purification steps. Temperature and pH ofthe treatment are not critical as far as they are not detrimental to theprotease. The enzyme compositions, which can be treated with the methodof the invention, are derived from animal organs (e.g. crude animalorgan extracts) or bacteria like Bacillus subtilis or licheniformis(e.g. fermentation broths). With the method of the invention more than80% of the amylase is inactivated leaving the protease activity morethan 80% intact.

U.S. Pat. No. 5,139,943 (1992) describes a method for selective recoveryof microbially produced chymosin from mixtures of polypeptides orenzymes produced by fermentation, like for example α-amylase. The methodof the invention is based on the use of a two phase, liquid-liquidsystem having partition coefficient for chymosin greater than about 85.The two-phase system is obtained by adding PEG and a salt like aphosphate or a sulphate to the aqueous enzyme composition. The chymosinis selectively extracted into the organic phase while the amylase staysin the water phase. Chymosin can be recovered from the PEG via ionexchange chromatography. By using a pH lower than 3, partitioncoefficients for chymosin can be obtained of about 1000, allowing fullseparation from amylase and a chymosin recovery of about 96-98%.

International Patent Application WO 97/20921 (published Jun. 12, 1997)describes a method for the selective inactivation of at least oneundesirable enzyme from an enzyme mixture comprising desirable andundesirable enzymes. The enzyme mixture is treated at pH lower than 5and at a temperature from 2 to 75° C. for at least 20 seconds, and/or atpH higher than 9 and at a temperature from 2 to 75° C. for at least 20seconds. By this method amylase is completely inactivated by treating anenzyme mixture comprising amylase and cellulase at pH 3.5 for 1 min at70° C., while 96% of the cellulase activity remains intact (Example 4).On the other hand only 2% of the protease activity is left when treatingan enzyme mixture comprising lipase and protease at pH 3.5, 45° C. for60 minutes. In mixtures comprising cellulase and protease (Example 7),the protease is completely inactivated by treating at 3° C. or 25° C.for 60 minutes and at a pH of 2.5.

Some methods described in the art have the disadvantage of being ratherlaborious. In general the methods in the art are characterised by a lowselectivity towards inactivation of amylase and by a relatively highloss in protease activity.

During the microbial production of chymosin and Rhizomucor mieheiaspartic protease via fermentation, the enzyme amylase is also producedin the fermentation broth. The dairy farming industry is now striving toobtain higher cheese yields and dairy products of superior quality. Thelatter highly depends upon the purity of the milk-clotting enzymesemployed in the production of these products. One of the problemsconnected with the presence of amylase in milk-clotting enzymes are theunwanted side activities of amylase in the whey, which can bedetrimental to further applications of the whey. Therefore one purposeof the invention is to provide a simple and efficient method toinactivate amylase in the presence of Rhizomucor miehei asparticprotease with high selectivity and with high recovery of the protease.

Surprisingly it has been found that the inactivation of amylase in thepresence of Rhizomucor miehei aspartic protease can be achieved by thepresent invention, which comprises keeping the Rhizomucor mieheiaspartic protease solution comprising amylase at a pH between 2.1 and2.8 and at a temperature between 20° C. and 40° C. for a period of timewhich is sufficient to inactivate amylase for at least 95%. Employingthese conditions results in an unexpected increase in amylasedeactivation whilst maintaining a high level of aspartic proteaseactivity.

Preferably, the solution is kept at this pH and temperature for a periodof time between 0.5 and 24 hours.

Advantages of this method are the simplicity and especially the highselectivity with which amylase is inactivated in Rhizomucor mieheiaspartic protease solutions comprising amylase. It has been surprisinglyfound that with the method of the invention at least 90%, preferably atleast 95% of aspartic protease activity is maintained in the Rhizomucormiehei aspartic protease solution after treatment while the residualamylase activity is generally lower than 0.05 RAU/g, preferably lowerthan 0.001 RAU/g, more preferably lower than 0.0005 RAU/g.

With the term “amylase” it is intended any enzyme with amylase activity,for example α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), γ-amylase(EC 3.2.1.3), amylase III (2.4.1.161) or any mixture thereof.

1 RAU (Amylase activity Unit) is defined as the amount of enzyme thatwill convert under standardized conditions (pH=6.6, 30° C.) 1 mg solublestarch per minute. The IMCU (International Milk Clotting Unit) isdefined for bovine rennets by the International Dairy Federation (IDF),protocol 157: 1992.

With the term “Rhizomucor miehei aspartic protease” it is intended theaspartic protease homologously produced in Rhizomucor miehei. A processfor the preparation of the enzyme via fermentation is described in theU.S. Pat. No. 3,988,207. With the term “Rhizomucor miehei asparticprotease” it is also intended to encompass a recombinant Rhizomucormiehei aspartic protease, i.e. a Rhizomucor miehei aspartic proteaseproduced in a host organism transformed with DNA coding for theRhizomucor miehei aspartic protease. The host organism can be a fungus,e.g. a yeast or a filamentous fungus. Preferably the host organism is afilamentous fungus selected from the genera of Aspergillus, Trichoderma,Penicillium, Fusarium or Humicola. Most preferably, the filamentousfungus belongs to the genera Aspergillus or Trichoderma. The use ofAspergillus niger, Aspergillus nidulans or Aspergillus oryzae as a hoststrain is preferred. Method for the production of a recombinantRhizomucor miehei aspartic protease in a host organism is described inEuropean patent EP0700253B1.

With the term “Rhizomucor miehei aspartic protease solution” it isintended a solution, preferably an aqueous solution, comprising amylaseand Rhizomucor miehei aspartic protease.

The method of the invention can be advantageously applied to Rhizomucormiehei aspartic protease derived from fermentation broths or derivativesthereof obtainable at any one of the stages during the down-streamprocess, generally before the formulation step. The Rhizomucor mieheiaspartic protease solution is preferably a fermentation broth, mostpreferably a fermentation broth derived from the fermentation ofRhizomucor miehei, which can be obtained according to methods known inthe art, for example like described in “Pilot plant experiment” of theU.S. Pat. No. 3,988,207. In another preferred embodiment of theinvention, the fermentation broth is a fermentation broth of a hostorganism transformed with DNA encoding the Rhizomucor miehei asparticprotease which organism expresses the protease. Generally, theRhizomucor miehei aspartic protease solution is obtained from thefermentation broth after removal of the cells. The latter may beachieved by killing of the microorganisms in the fermentation broth byone of the several methods known in the art and by removal of the celldebris. Removal of the cells can be achieved by one or more solid/liquidseparation techniques like flocculation, centrifugation, filtration, andmembrane separation. Generally, the Rhizomucor miehei aspartic proteasesolution is obtained from the fermentation broth after removal of thecells and concentration of the cell free solution prior use in themethod of the invention. Concentration may be achieved by evaporation ormembrane concentration; preferably the membrane concentration isachieved by ultrafiltration techniques. Therefore in a preferredembodiment of the invention the Rhizomucor miehei aspartic proteasesolution is a cell-free and/or concentrated fermentation broth. With“cell-free” it is intended free of any particle or cell with diameter of0.4 μm or higher.

It has been observed that the addition of an agent such as sodiumchloride to the Rhizomucor miehei aspartic protease solution stabilisesthe Rhizomucor miehei aspartic protease decreases the loss in asparticprotease activity during the method of the invention. Examples of theseagents are sodium chloride, potassium chloride, glycerol, sorbitol,polyethylene glycol. In a preferred embodiment of the invention, theRhizomucor miehei aspartic protease solution comprises an agent havingstabilising effect on Rhizomucor miehei aspartic protease. Mostpreferably the Rhizomucor miehei aspartic protease solution comprisessodium chloride. When sodium chloride is present, this is generallyadded to the fermentation broth in an amount comprised between 50-200g/kg.

The pH of the Rhizomucor miehei aspartic protease solution used in themethod of the invention can be adjusted using food acceptable acids orbases well within the knowledge of those skilled in the art. Adjustmentof the pH can be effected before during or after the enzyme mixture hasreached the heating temperature. However the heating time should startonce the solution has the correct pH. In a preferred embodiment of theinvention adjustment of the pH of the enzyme mixture is achieved priorto heating the enzyme mixture.

It has been observed that the best results in maintenance of proteaseactivity and inactivation of amylase are obtained when, in a method ofthe invention, the Rhizomucor miehei aspartic protease solution is keptat a pH between 2.3 and 2.6 and/or kept at a temperature between 32° C.and 38° C. Preferably the solution is kept at this pH and temperaturefor a period of time between 0.5 and 24 hours. Therefore, in a preferredembodiment of the invention the pH of the Rhizomucor miehei asparticprotease solution during the inactivation step is between 2.3 and 2.6.In another preferred embodiment the temperature at which the solution iskept during the inactivation step is comprised between 32° C. and 38° C.In a third preferred embodiment the period of time at which the reactionis kept at the desired temperature is between 4 and 12 hours.

Once the amylase inactivation step has been completed, the temperatureof the solution is generally decreased to between about 4° C. to 25° C.,preferably to 4° C., and the pH of the solution is increased to a valueat which the Rhizomucor miehei aspartic protease is stable, usually at apH between about 4 and 6.

With the method of the invention, amylase is inactivated with highselectivity in Rhizomucor miehei aspartic protease solutions. At the endof the treatment at least 90%, preferably at least 95% of the asparticprotease activity is maintained while the residual amylase activity isgenerally lower than 0.05 RAU/g, preferably lower than 0.001 RAU/g, morepreferably lower than 0.0005 RAU/g. The level of amylase activity in theRhizomucor miehei aspartic protease solution before the inactivationstep is generally 100 RAU/g or higher. Therefore, the degree of amylaseinactivation in the solution after the treatment is generally at least99.95%.

It is known that Rhizomucor miehei aspartic protease is highly heatstable. This characteristic can be disadvantageous, as duringcheese-making manufacture the enzyme remains partially active in thewhey after pasteurisation, causing undesired milk clotting when the wheyis used in other preparations. Therefore the Rhizomucor miehei asparticprotease comprised in coagulating enzyme preparations is preferablymodified to increase its thermal destabilisation. In case the Rhizomucormiehei aspartic protease used in a method of the invention has not beentreated to increase its thermal destabilisation, the method of theinvention can comprise a step, to be applied after the amylaseinactivation, to increase the thermal destabilisation of the Rhizomucormiehei aspartic protease. The treatment to increase Rhizomucor mieheiaspartic protease thermal destabilisation can also be applied prior tothe amylase inactivation step. In this case the amylase inactivationstep is preferably applied at a temperature between 20° C. and 25° C.,preferably at about 20° C., the other parameters remaining the same. Theenzyme solution preferably comprises sodium chloride or an agent havinga stabilising effect on Rhizomucor miehei aspartic protease. When sodiumchloride is used, this is generally added to the fermentation broth inan amount between 50-200 g/kg.

In a preferred embodiment, after inactivation of amylase the thermaldestabilisation of the Rhizomucor miehei aspartic protease in thesolution is increased with a method known in the art.

The method used to increase the thermal destabilisation of Rhizomucormiehei aspartic protease is not critical and may comprise any of theseveral methods known in the art to perform this task. For example theenzyme product can be treated in an aqueous medium with an oxidisingagent containing active halogen, e.g. hypochlorite (e.g. the GB PatentNo. 2,045,772B). Another possibility is to treat the enzyme product inaqueous medium with an acylating reagent, e.g. acetic or propionicanhydride (e.g. the GB Patent No. 2,038,339B). Preferably the enzymeproduct is treated in aqueous medium with a peroxy acid like aninorganic peroxy acid or a lower aliphatic peroxy acid. Preferablyperoxy acetic or peroxy propionic acid is used (GB Patent No.2,045,773). The way to perform these methods is widely described in theliterature and therefore well within the knowledge of those skilled inthe art.

The invention also provides a Rhizomucor miehei aspartic proteaseobtainable by a method of the invention and further providescompositions comprising such aspartic proteases. The Rhizomucor mieheiaspartic protease comprises less than 0.0005 RAU amylase per 2000 IMCUprotease.

The enzyme product comprising Rhizomucor miehei aspartic proteaseobtainable by a method of the invention can be further processed.Following the amylase inactivation step or the increase in thermaldestabilisation of Rhizomucor miehei aspartic protease any conventionalmethod known in the art is suitable to prepare the enzyme for furtheruse.

The enzyme product can be used for the preparation of a food stuff.These compositions may comprise other enzymes. Therefore the inventionalso provides compositions comprising Rhizomucor miehei asparticprotease obtainable by a method of the invention. Such compositions maybe solid or liquid compositions. The solid compositions may be in theform of tablets, granules, powders, crystals etc.

The Rhizomucor miehei aspartic protease obtainable by a method of theinvention or alternatively the composition of the invention can be usedfor the production of a dairy product. Milk of different sources can beused in the production of such product, which preferably is a cheeseproduct. Examples of milk, which are obtainable by the method of theinvention, are cow milk, goat milk, sheep milk, camel milk etc. TheRhizomucor miehei aspartic protease is suitable for the production ofany type of cheese or dairy product. The use of microbial asparticproteases as clotting enzymes in cheese making is well documented in theart. An advantage of the Rhizomucor miehei aspartic protease obtainableby the method of the invention or alternatively the compositions of theinvention is the very low level or absence of amylase activity. The useof the enzyme of the invention or of the aforementioned composition as aclotting enzyme in cheese making allows, for example, a moreadvantageous further processing of the whey obtained after separation ofthe curd. This whey, being free of amylase activity, can be used withbetter results for the preparation of dairy products comprising starch,gluten, etc.

The invention will now be illustrated by way of examples, which howevershould not be considered as limiting.

EXAMPLE 1

The following example shows the results of amylase inactivation in aRhizomucor miehei cell-free, concentrated fermentation broth obtainedfrom a crude Rhizomucor miehei fermentation broth after the steps of: 1)solid-liquid separation, 2) polish filtration, and 3) ultrafiltrationcomprising 100 g/kg of NaCl as stabiliser. The Rhizomucor mieheiaspartic protease comprised in the fermentation broth has not beentreated to increase its thermal destabilisation. The amylase activityand the Rhizomucor miehei aspartic protease activity in the solution aremeasured with standard methods known to those skilled in the art.

TABLE 1 pH 2.5 3.0 Temperature (° C.) 20 30 40 Time (hours) 2 4 6 8 2 48 2 4 6 8 12 24 R. miehei Asp. 97 101 100 99 99 102 100 104 97 99 99 10298 Protease (%) Amylase (RAU/g) 218 100 30 10 25 5 0.01-1 150 70 26 6 <1<1

TABLE 2 pH 2.5 Temperature (° C.) 35 40 Time (hours) 4 8 4 8 R. mieheiAsp. prot. 101 100 100 96 (%) Amylase (RAU/g) <0.0005 <0.0005 <0.0005<0.0005 Note: Amylase activities of 1 RAU/g or higher are measured viaUV-Vis absorption. Activities lower than 1 RAU/g are measured via aPetri dish method. The detection limit is 0.0005 RAU/g.

Table 1 shows the residual amylase and the residual aspartic proteaseactivity after treatment, the latter effected at a pH between 2.5 and3.0, a temperature between 20° C. and 40° C., for a time between 2 and24 hours, on a cell-free, concentrated fermentation broth comprising3950 IMCU/g R. miehei aspartic protease activity and 520 RAU/g amylaseactivity

Table 2 shows the same values at a pH of 2.5, a temperature between 30°C. and 40° C., for a time between 4 and 8 hours, on a cell-free,concentrated fermentation broth comprising 2000 IMCU/g R. mieheiaspartic protease activity and 250 RAU/g amylase activity.

These experiments show that to obtain optimal amylase inactivation (i.e.at least 99.99% amylase inactivation) in Rhizomucor miehei, cell-free,concentrated fermentation broth comprising Rhizomucor miehei asparticprotease which has not been treated to increase its thermaldestabilisation, with minimal losses in aspartic protease activity, thefollowing conditions can be applied: a pH of about 2.5, a temperature ofabout 35° C. and a heating time of about 4 hours.

EXAMPLE 2

This example shows the results of amylase inactivation treatmenteffected on Rhizomucor miehei cell-free, concentrated fermentation brothprestabilised with 100 g/kg of NaCl.

TABLE 3 Batch 1 2 3 R. miehei R. miehei R. miehei Amylase Asp. AmylaseAsp. Amylase Asp. Time (h) (RAU/g) Prot. (%) (RAU/g) Prot. (%) (RAU/g)Prot. (%) 0 225 100 490 100  550 100  4 <0.0005 101 0.001-0.005 98 Nd Nd5 Nd Nd 0.0005-0.001  98 Nd Nd 6 Nd Nd <0.0005 96 0.0005 98 8 <0.0005100 Nd Nd Nd Nd Note: Amylase activities of 1 RAU/g or higher aremeasured via UV-Vis absorption. Activities lower than 1 are measured viaa Petri dish method. The detection limit is 0.0005 RAU/g. Nd = notdetermined.

Table 3 shows the residual amylase and the residual aspartic proteaseactivity after treatment effected at pH 2.5, a temperature of 35° C. forbetween 0 and 8 hours. Batch 1 comprises 2000 IMCU/g R. miehei asparticprotease, 250 RAU/g amylase, batch 2 comprises 3990 IMCU/g R. mieheiaspartic protease, 626 RAU/g amylase and batch 3 comprises 3389 IMCU/gR. miehei aspartic protease, 701 RAU/g amylase.

These results show that inactivation of amylase in Rhizomucor mieheicell-free, concentrated fermentation broth derivatives comprising sodiumchloride and Rhizomucor miehei aspartic protease which has not beentreated to increase its thermal destabilisation, can be achieved within4 hours with average aspartic protease activity losses lower than 5%.

EXAMPLE 3

The following example shows the results of amylase inactivation inRhizomucor miehei cell-free, concentrated fermentation broths comprisingRhizomucor miehei aspartic protease, which has been treated to increaseits thermal destabilisation. The concentrates have been stabilised with100 g/kg of sodium chloride. The results of the experiments, expressedas residual amylase activity and residual Rhizomucor miehei asparticprotease activity after the inactivation step are shown in Table 4. Allexperiments have been executed at 20° C., at a pH between 2.3 and 2.7,for a time of 0 to 8 hours.

TABLE 4 Assay 1 Assay 2 Assay 3 Time pH 2.5 pH 2.3 pH 2.7 (h) Amyl RAFAmyl RAF Amyl RAF 0 125 100 125 100 125 100 6 0.018 97 0.013 95 0.029 968 0.018 99 0.013 94 0.027 99 Note: Amyl: residual amylase activity(RAU/g); RAF: percentage of residual Rhizomucor miehei aspartic proteaseactivity. Amylase activities of 1 RAU/g or higher are measured via UV-Vis absorption. Activities lower than 1 are measured via a Petri dishmethod. The detection limit is 0.0005 RAU/g.

These results show that with Rhizomucor miehei solutions comprisingRhizomucor miehei aspartic protease, which has been treated to increaseits thermal stabilisation, higher aspartic protease activity losses andhigher levels of residual amylase activity can be observed. However thelosses in protease activity are always lower than 10%, and the residualamylase activity is always lower than 0.05 RAU/g (i.e. the degree ofamylase inactivation is 99.98%).

1. A method to inactivate amylase in a solution containing an asparticprotease from Rhizomucor miehei and said amylase, which method comprisesincubating said solution at a pH between 2.1 and 2.8 and at atemperature between 20° C. and 40° C. for a period of time sufficient toinactivate at least 95% of amylase activity; wherein residual proteaseactivity in the solution at the end of the incubation is at least 90% ofprotease activity at the beginning of the incubation.
 2. The method ofclaim 1, wherein said time is between 0.5 and 24 hours.
 3. The method ofclaim 1, wherein residual amylase activity is 0.05 RAU/g or lower. 4.The method of claim 1, wherein the pH is between 2.3 and 2.6.
 5. Themethod of claim 1, wherein the temperature is between 32° C. and 38° C.6. The method of claim 2, wherein said time is between 4 and 12 hours.7. The method of claim 1, wherein the solution is a cell-free and/orconcentrated fermentation broth.
 8. The method of claim 7, wherein thefermentation broth is from a filamentous fungus selected from the groupconsisting of Aspergillus, Trichoderma, Penicillium, Fusarium, andHumicola.
 9. The method of claim 1, which further comprises increasingthermal destabilisation of the aspartic protease.
 10. The method ofclaim 1, which further comprises increasing thermal destabilisation ofthe aspartic protease prior to said incubation step.
 11. The method ofclaim 10, wherein said inactivating step is carried out at a temperaturebetween 20° C. and 25° C.
 12. A protease composition comprising anaspartic protease from Rhizomucor miehei, wherein said compositioncomprises less than 0.001 RAU of amylase per 2000 IMCU of protease. 13.A second composition comprising the protease composition of claim 12.14. The second composition of claim 13, which is a solid or a liquidcomposition.
 15. The second composition of claim 14, which is in theform of a tablet, a powder or a granule.
 16. A method to produce a dairyproduct which method comprises coagulating a dairy product preparationby contacting said preparation with the protease composition of claim 12or a second composition thereof.
 17. The method of claim 16, wherein thedairy product is a cheese product.
 18. The composition which isobtainable by the method of claim
 1. 19. A method to inactivate amylasein a solution containing (i) an aspartic protease from Rhizomucormiehei, (ii) a stabilizing agent for the aspartic protease, and (iii)said amylase, which method comprises incubating said solution at a pHbetween 2.1 and 2.8 and at a temperature between 20° C. and 40° C. for aperiod of time sufficient to inactivate at least 95% of amylaseactivity; wherein residual protease activity in the solution at the endof the incubation is at least 90% of protease activity at the beginningof the incubation.
 20. The method of claim 19, wherein said time isbetween 0.5 and 24 hours.
 21. The method of claim 20, wherein said timeis between 4 and 12 hours.
 22. The method of claim 19, wherein residualamylase activity is 0.05 RAU/g or lower.
 23. The method of claim 19,wherein the pH is between 2.3 and 2.6.
 24. The method of claim 19,wherein the temperature is between 32° C. and 38° C.
 25. The method ofclaim 19, wherein the solution is a cell-free and/or concentratedfermentation broth.
 26. The method of claim 25, wherein the fermentationbroth is from a filamentous fungus selected from the group consisting ofAspergillus, Trichoderma, Penicillium, Fusarium, and Humicola.
 27. Themethod of claim 19, wherein the stabilising agent is sodium chloride.28. The method of claim 19, which further comprises increasing thermaldestabilisation of the aspartic protease.
 29. The method of claim 19,which further comprises increasing thermal destabilisation of theaspartic protease prior to said incubation step.
 30. The method of claim29, wherein said inactivating step is carried out at a temperaturebetween 20° C. and 25° C.
 31. A protease composition comprising anaspartic protease from Rhizomucor miehei and a stabilising agent for theaspartic protease, wherein said composition comprises less than 0.001RAU of amylase per 2000 IMCU of protease.
 32. A second compositioncomprising the protease composition of claim
 31. 33. The secondcomposition of claim 32, which is a solid or a liquid composition. 34.The second composition of claim 33, which is in the form of a tablet, apowder or a granule.
 35. The composition of claim 31, wherein thestabilising agent is sodium chloride.
 36. The composition which isobtainable by the method of claim
 19. 37. A method to produce a dairyproduct which method comprises coagulating a dairy product preparationby contacting said preparation with the protease composition of claim 31or a second composition thereof.
 38. The method of claim 37, wherein thedairy product is a cheese product.